Apparatus for performing saturation analyses

ABSTRACT

The invention relates to an apparatus which may be used in saturation determinations. The instrument comprises a reaction compartment and a separating compartment separated from the reaction compartment. The reaction compartment is provided with a wall portion which may be rendered permeable to liquid, thereby enabling the contents of the reaction compartment to flow to the separating compartment.

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l l NOV. 11, 1975 Arlman [54l APPARATUS FOR PERFORNHNG l.'/6579-1 (i/l93l) Hirth a a i r l a v a v i 128/172 SATURATION ANALYSES 2.612163 9/1952 Normanmm 215/6 3.380.888 4/l9o8 Numerof et al. 23/230 B [75] Jacob Arlman P- 3.659.104 4/197: Gross n n 23/330 8 x Netherlands 1707.353 12/1972 Kuhiatowictnm. 23/130 B [73} Assignee: U.S. Philips Corporation. New

York- NY. PIT/HUI) Emminer-Joseph Scovronek V c Assistant [:'.\zm2iricr-Dale Lovercheck {n1 Fllcd' Sept Alwrm'y. Agent. or Firm-Frank R. Trifari; Norman {21] Appl, No.: 290.650 N. Spain {301 Foreign Appllcatlon PllOl'll) Data I 57} ABSTRACT Sept 2 l. l97l Netherlands .7 7l l2927 The invention relates to an apparatus which ma) be I52] U.S. Cl. t. 23/253 R; 23/230 8123/2591 used in saturation determinations The instrument 215/6 comprises a reaction compartment and a separating (51] Int. Cl.' GOIN 31/06: B0lL 3/00 compartment separated from the reaction compart- [58] Field of Search 23/230 R. 253 R. 230 B. ment. The reaction compartment is provided with a 23/259. 292, 258.5; 206/47 A; 2 lS/f); 128/272 wall portion which may be rendered permeable to liquid thereby enabling the contents of the reaction [56] References Cited compartment to flow to the separating compartment.

UNlTED STATES PATENTS M06334 3/1929 Toch .4 206/47 A 3 Chums 6 Drawmg figures III/III [Ill U.S. Patent N0v.11, 1975 SheetlofZ 3,918,909

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APPARATUS FOR PERFORMING SATURATION ANALYSES The invention relates to an apparatus for use in performing saturation analyses. According to the principle of a saturation analysis, a quantity of a chemical or biological substance to be determined is mixed in the presence of a binder with a known quantity of the same substance which, however, is radioactively labelled.

An equilibrium reaction is produced between the binder, the substance to be determined and the radioactive substance, in which reaction molecules of the substance to be determined and of the radioactive substance are bound to the binder and at the same time bound molecules both of the substance to be determined and of the radioactive substance are released. The degree in which each of the two substances is bound to the binder depends upon the concentration ratio of the two compounds. A binder used in such a reaction is sometimes referred to as a competitive binder. After an equilibrium condition has been reached between the binder, the substance to be determined and the radioactive substance, the extent to which the radioactive substance has been bound to the binder is ascertained. For this purpose the bound radioactive substance is separated from the unbound form and subsequently the amount of the bound or of the free radioactive compound is determined by measuring the radioactivity. The percentage of the free or bound radioactive substance is a measure of the quantity to be determined of the non-radioactive compound. The exact quantity of the non-radioactive compound may be derived from the percentage found by means of standard data.

The aforementioned separation between the bound and unbound radioactive compounds may be effected in various manners. Thus, for example, the desired separation may be performed by means of a molecular sieve or a selective adsorbent which binds or stops only one of the two structures of the radioactive compound, either the bound one or the free one, and consequently releases the other structure.

On the basis of the principle of saturation analysis various hormonal and non-hormonal substances such, for example, as triiodothyronine, thyroxin, vitamin B12 and insulin may be determined. A special type of saturation analysis is the radio-immunodetermination. In this determination a specific antibody is used as a competitive binder. In this manner, for example, insulin is determined by means of a radioimmuno-investigation.

The invention relates in particular to an apparatus for performing a saturation analysis as described hereinbefore which includes a tubular member which is open at both ends and in its lower part contains a separating agent and in its upper part has a reaction compartment.

Such an apparatus is used, for example, in the thyroid function test known under the trade name TrilutefThe thyroid function test is based on the fact that the triiodothyronine produced by the thyroid is bound to specific serum albumins, the degree of occupation of the albumin by triiodothyronine being a measure of the thyroid activity.

In a thyroid function test a controlled amount of radio-active triiodothyronine is added to the serum albumin, the free binding sites still present in the serum al bumin being occupied by molecules of the radioactive substance. Subsequently the free triiodothyronine is separated from the triiodothyronine bound to the serum albumin by means of a specific adsorbent, such as Sephadex, after which the quantity of radioactive triiodothyronine which is not bound to the serum albumin and is retained by the Sephadex is determined, The result is a measure of the binding capacity of the serum albumin investigated and enables the degree of occupation of the serum albumin by the thyroid hormone to be derived, which gives an impression of the thyroid function,

The Trilute test proceeds as follows: seven drops of radioactive triiodothyronine and 0.05 ml of serum are successively applied to a Sephadex column which is disposed in the lower part of a tubular member which is open at both ends and the lower opening of which is closed by a protective cap. The protective cap is removed, the liquid present under the Sephadex column is discharged and the radioactivity of the column is measured. Subsequently the column is washed with 3 ml of water, so that the triiodothyronine bound to albumin is removed, whereupon the radio-activity is again determined. The amount of non-radioactive triiodothyronine can be derived by means of standard data from the ratio between the two measurements.

The instrument used in the known Trilute test has the disadvantage that an equilibrium between serum albumin and added radioactive triiodothyronine is to be established in the presence of the separating agent, in this case the Sephadex column. Establishment of an equilibrium in a heterogeneous medium takes much time and gives rise to inaccuracy, for it is not unlikely at all that, before the said equilibrium has been established, the Sephadex should retain free triiodothyronine molecules, thereby impeding correct establishment of the equilibrium. A further disadvantage of the known test is the comparatively large number of operations to be performed, which is to be avoided especially with radioactive substances. Another disadvantage consists in that in each test at least two determinations of the radioactivity have to be carried out.

We have now developed an apparatus of the type described at the beginning of this specification which does not have the aforementioned disadvantages. The apparatus according to the invention is characterized in that the reaction compartment is a container which is closed with respect to the separating agent and a wall portion of which facing the separating agent may be brought into a liquid-pe rrneable form. The presence of the reaction compartment from which the separating agent is excluded in the apparatus according to the invention has the advantage that the reactions which in the operation of the apparatus according to the invention take place in the compartment proceed in a homogeneous medium. Consequently the required equilibrium between the reagents in the reaction container, ie between the substance to be determined, the radioactive susbstance and the competitive binder, is established rapidly and accurately. The separating agent remains entirely outside the reaction container and does not influence the reactions carried out in this container.

The closed reaction container provides the further advantage that the radioactive substance and, if desired, the binder as well, which two substances are necessary in the saturation analysis to be carried out in the apparatus according to the invention, may previously be introduced into the container during manufacture.

Thus the user of the instrument need only to add the sample to be investigated to the reagents already present in the container. This ensures that manipulations of radioactive substances by the user are reduced to a minimum. Furthermore, during manufacture the amount of radioactivity introduced into the reaction container can accurately be determined and be equal for each reaction container, so that the user need only perform a single measurement of the initial radioactivity, which may then be used as a standard for all determinations.

To enable the user to simply introduce a sample of the substance to be determined into the reaction container, the upper end of the container may be provided with a detachable cover. As an alternative, the upper surface of the container may be provided with a membrane made of a material which may readily be puncturcd with a sharp object. Suitable materials are, for example, rubber or a synthetic material. A sample of the substance to be determined may be aspirated into, for example, a hypodermic syringe, after which the needle of the syringe may be made to pierce the said membrane, enabling the syringe to be discharged into the reaction container.

The reaction compartment may be provided asa separate object in the tubular member, the opening of the member being closed by the upper surface of the reaction compartment.

In a preferred embodiment of the apparatus according to the invention the container forms part of the tubular member. An upper part of the tubular member then also is a wall part of the reaction compartment. The bottom of the reaction compartment is constituted by a wall which extends within the tubular member in a direction at right angles to the axis of this member and throughout its circumference engages the inner wall of the tubular member with a tight fit. The upper surface of the reaction compartment is formed by a wall which closes the opening in the tubular member, for example, a cover detachably placed on the end of the tubular member.

According to the invention a container wall facing the separating agent may be rendered permeable to liquid.

The feature of providing the container with a wall which is constructed so as to be capable of becoming permeable to liquid is essential for an understanding of the invention. This feature may be implemented in several forms in practice. For example, the wall part of the reaction container which faces the separating agent may contain a porous hydrophobic or lipophobic filter. A porous hydrophobic filter is used when the media in the reaction compartment and in the separating columns are hydrophilic. The porous hydrophobic filter prevents the hydrophilic liquid contained in the reaction compartment from penetrating through the filter into the separating agent. Only when by violent shaking of the apparatus according to the invention the air retained by the hydrophobic filter is expelled can liquid flow from the reaction compartment to the separating agent. The filter may be rendered permeable to liquid not only by shaking but also by producing an excess pressure either in the reaction compartment or in the separating column.

Another highly advantageous embodiment of the reaction compartment in the apparatus according to the invention consists in that the bottom of the reaction container is constituted, at least partly, by a pierceable membrane or partition.

A suitable material for such a membrane or partition is a synthetic material, a metal foil or the like.

In a further advantageous embodiment of the apparatus according to the invention the wall of the reaction container which faces the separating agent consists of a layer of a wax having a melting point between 40C and C. An example of a suitable wax is paraffin having a melting point of 60C. Such a wax wall may simply be provided by dripping the wax, which is heated and hence is liquid, on the separating agent present in the tubular member, the wax being subsequently solidified by cooling.

In still another embodiment of the apparatus according to the invention the upper and lower parts of the tubular member are joined to one another by an intermediate part which is provided with a central bore in which a slide is mounted which is displaceable in a direction at right angles to the axis of the bore so as to completely close the bore in one extreme position and to completely open the bore in the other extreme position.

The invention will now be described more fully with reference to the accompanying diagrammatic drawings, without being restricted to the embodiments shown.

FIGS. 1 to 5 show schematically various embodiments of the apparatus according to the invention.

FIG. 1 shows an embodiment in which the reaction compartment is in the form of a separate unit.

FIGS. 2, 3, 4 and 5 shown embodiments in which the upper part of the tubular member also is a wall portion of the reaction compartment.

FIG. 5 shows a construction in which the upper and lower parts of the tubular member are joined by an intermediate piece.

FIG. 6 shows a modification of the hollow cylindrical downward projection shown in FIGS. 1 to 5.

Referring now to FIGS. 1 to 5, reference numeral 1 denotes a tubular member open at both ends. At the lower end the member I is provided with a hollow cylindrical downward projection in which a porous plug 6 has been inserted. The projection 2 is provided with a detachably secured closing cap 7. A separating agent 8 is disposed in the lower part of the tubular member 1. If the apparatus according to the invention is used to determine triiodothyronine, the separating agent 8 may consist of Sephadex taken up in a liquid 9. The upper part of the tubular member 1 contains a reaction compartment 10, various embodiments of which are shown in FIGS. 1 to 5.

The reaction compartment 10 shown in FIG. 1 has a tubular part 13 which at the upper end is provided with a rim 14 to which a cover 11 is detachably secured.- The lower end of the tubular part 13 has a bottom 12 in which a central, preferably conical, aperture 15 has been formed. The bottom 12 is provided with a membrane 16 which is made of a synthetic material, a metal foil or a similar readily pierceable material. The membrane 16 may be cemented to the inner surface of the bottom l2 and entirely closed the opening IS in the bottom 12. The outer surface of the part 13 engages the inner wall of the member 1. A suitable choice of the materials from which the part 13 and the member 1 are made, for example the use of a synthetic material for the part 13 and glass for the member I, ensures a satisfactory seal between the part I3 and the member I. As an alternative, the outer diameter of the part 13 may be slightly smaller than the inner diameter of the member I, a sealing ring made, for example, of nylon being interposed between them.

When, for example, the binding capacity of serum albumin for triidothyronine is to be determined, the apparatus shown in FIG. 1 is used in the following manner.

The reaction compartment I0 contains a known quantity of radioactive triiodothyronine which has been introduced during manufacture. To perform a triiodothyronine determination the cover II is removed and serum is added to the contents of the compartment 10. The serum containing the triiodothyronine may, if desired, be introduced into the compartment without the cover 11 being removed in that, for example, the serum is drawn into a hypodermic syringe, the needle is inserted through the cover II and the contents of the syringe are injected into the compartment 10 through the needle. In the reaction compartment an equilibrium between the reagents is rapidly established. Then the cap 7 is removed and the membrane I6 is pierced at the area of the opening IS in the bottom 12, for example, by means of a glass rod. Owing to the conical shape of the opening the tool used to pierce the membrane cannot penetrate into the space immediately below the bottom 12. Under the influence of gravity the equilibrium mixture contained in the compartment 10 moves into the separating agent 8, which comprises Sephadex taken up in a liquid 9. The triiodothyronine which is not bound to serum albumin and hence is free will then become bound to the sephadex. Subsequently washing liquid is introduced into the reaction compartment and flow through the Sephadex, carrying along the triiodothyronine bound to albumin and not retained by the Sephadex. The washing liquid is discharged from the apparatus according to the invention through the projection 2. Subsequently the radioactivity of the radioactive triiodothyronine left in the Sephadex column is measured. Fromthe ratio between the measured radioactivity initially present in the reaction compartment 10 the binding capacity of the serum albumin for the triiodothyronine may be deduced by means of standard data. It should be pointed out that the movement of the equilibrium mixture from the reaction compartment 10 into the separating agent 8 and the subsequent treatment with washing liquid may be accelerated by creating a partial vacuum in the lower part of the apparatus according to the invention. For example, several apparatuses according to the invention may be connected in parallel to a vacuum pipe. This provides an interesting possibility of simultaneously performing several determinations. Moreover the washing liquid which contains the radioactive constituents from several apparatuses may be safely conveyed away.

FIG. 2 shows an embodiment of an instrument according to the invention in which a lateral wall 18 of the reaction container 10 forms part of the tubular member 1. The bottom of the reaction container 10 is a membrane 16 which is made of a synthetic material, a metal foil or a similar readily pierceable material and is clamped between rings 19 which may be made of ny- Ion.

In FIGS. 3 and 4 the upper part 18 of the tubular member I also is a wall part of the reaction container 10. The bottom of the reaction container is constituted by a porous hydrophobic filter 17 which is clamped between rings 19 and may be made of, for example, a

foamed synthetic material. The Sephadex 8 taken up in 5 the liquid 9 extends up to the lower surface of the hydrophobic filter 17 (FIG. 3).

In FIG. 4 the bottom of the reaction container 10 consists of a layer of a wax 28 which has a melting point between 40C and 100C, for example a layer of paraffin. Such a wax layer is provided by dripping wax heated to a temperature above its melting point on the Sephadex 8 and then solidifying the wax by cooling.

In the embodiment shown in FIG. 5 the tubular member 1 comprises two parts which are joined to one another by an intermediate piece 20. The upper part I8 of the tubular member 1 constituted the lateral wall of the reaction compartment 10 and is provided with a detachable cover 11. The upper surface of the intermediate piece 20 also is the bottom of the reaction compartment 10. The intermediate piece 20 comprises a ring 22 provided with a central bore 21. The ring 22, which may be made of a synthetic material, has a slot 23 formed in it which extends in a direction at right angles to the bore 21 and in which a slide 24 fits which is movable in the said direction. The slide is provided at one end with a handle 25 which protrudes from the ring 22 and at the other end with a stop 26. The stop 26 tits in a recess 27 in the ring 22. The slide 24 can be displaced in the slot 23 by means of the handle 25 in such a manner that in an extreme inner position of the slide 24 the bore 21 is entirely closed. In this position the stop 26 is located in the recess 27. In the other extreme position of the slide the bore 21 is entirely uncovered and the stop 26 engages the inner wall of the ring 22.

FIG. 6 shows a modified embodiment of the hollow cylindrical downward projection 2 shown in FIGS. 1 to 5. In FIG. 6 reference numeral 3 denotes a spout-like member provided with a flange 4. The flange 4 engages the inner surface of the re-entrant end 5 of the tubular member I. The flange 4 may be cemented to the end 5. As an alternative, the flange 4 may be urged into engagement with the end 5 by means of a ring.

The operation of the apparatus shown in FIGS. 2 to 5 corresponds to that of the embodiment shown in FIG. 1. The manner in which the bottom of the reaction compartment 10 is made permeable to liquid is different in the different embodiments.

In the apparatuses shown in FIGS. 2 and 4 the bottom is made permeable in that the membrane 16 and the wax layer 28 respectively are pierced by a possibly sharp object. In the apparatus shown in FIG. 3 the filter is made permeable to liquid by shaking the apparatus, or by producing an excess pressure in the reaction compartment or a reduced pressure in the compartment containing the separating agent. In the apparatus shown in FIG. 5 the contents of the reaction compartment are permitted to move towards the separating agent by the withdrawal of the slide.

What is claimed is:

l. A device suitable for performing a saturation analysis in which by the use of a separating agent a radioactive-substance bound to a competitive binder is separated from its unbound form which device comprises a tubular member open at both ends and containing in a lower part the separating agent and containing in an upper part a reaction compartment containing a radioactive reagent, said reaction compartment being a container closed with respect to the separating agent and having a non-porous bottom portion facing the separating agent which may be rendered permeable to liquid and said non-porous bottom portion including a hydrophobic porous filter capable of being made permeable to liquid by shaking or by providing a pressure differential between the reaction compartment and the remainder of the tubular member.

2. A device suitable for performing a saturation analysis in which by the use of a separating agent a radioactive-substance bound to a competitive binder is separated from its unbound form which device comprises a tubular member open at both ends and containing in a lower part the separating agent and containing in an upper part a reaction compartment, said reaction compartment being a container closed with respect to the separating agent and having a non-porous bottom portion facing the separating agent which may be rendered permeable to liquid and said non-porous bottom portion being constituted by a layer of wax the melting point of which lies between 40C and C.

3. A device suitable for performing a saturation analysis in which by use of a separating agent a radioactivesubstance bound to a competitive binder is separated from its unbound form which device comprises a tubular member open at both ends and containing in a lower part the separating agent and in an upper part is provided with a reaction compartment, the upper pan and the lower part of the tubular member being joined to one another by an intermediate member provided with a central bore and in which a slide is mounted displaceable in a direction at right angles to the axis of the bore so as to entirely close the bore in one extreme position and to entirely open the bore in the other extreme position. 

1. A DEVICE SUITABLE FOR PERFORMING A SATURATION ANALYSIS IN WHICH BY THE USE OF A SEPARATING AGENT A RADIOACTIVE-SUBSTANCE BOUND TO A COMPETIVE BINDER IS SEPARATED FROM ITS UNBOUND FORM WHICH DEVICE COMPRISES A TUBULAR MEMBER OPEN AT BOTH ENDS AND CONTAINING IN A LOWER PART THE SEPARATING AG*NT AND CONTAINING IN AN UPPER PART A REACTION COMPARTMENT CONTAINING A RADIOACTIVE REAGENT, SAID REACTION COMPARTMENT BEING A CONTAINER CLOSED WITH RESPECT TO THE SEPARETING AGENT AND HAVING A NON-POROUS BOTTOM PORTION FACING THE SEPARATING AGENT WHICH MAY BE RENDERED PERMEABLE TO LIQUID AND SAID NON-POROUS BOTTOM PORTION INCLUDING A HYDROPHOBIC POROUS FILTER CAPABLE OF BEING MADE PERMEABLE TO LOQUID BY SHAKING OR BY PROVIDING A PRESSURE DIFFERENTIAL BETWEEN THE REACTION COMPARTMENT AND THE REMAINDER OF THE TUBULAR MEMBER.
 2. A device suitable for performing a saturation analysis in which by the use of a separating agent a radioactive-substance bound to a competitive binder is separated from its unbound form which device comprises a tubular member open at both ends and containing in a lower part the separating agent and containing in an upper part a reaction compartment, said reaction compartment being a container closed with respect to the separating agent and having a non-porous bottom portion facing the separating agent which may be rendered permeable to liquid and said non-porous bottom portion being constituted by a layer of wax the melting point of which lies between 40*C and 100*C.
 3. A device suitable for performing a saturation analysis in which by use of a separating agent a radioactive-substance bound to a competitive binder is separated from its unbound form which device comprises a tubular member open at both ends and containing in a lower part the separating agent and in an upper part is provided with a reaction compartment, the upper part and the lower part of the tubular member being joined to one another by an intermediate member provided with a central bore and in which a slide is mounted displaceable in a direction at right angles to the axis of the bore so as to entirely close the bore in one extreme position and to entirely open the bore in the other extreme position. 